PCR Extension Time Calculator

Calculate recommended PCR extension times from your fragment length polymerase speed and GC content adjustments. See per cycle timing and total runtime estimates with safety margin suggestions. Supports fast enzymes and long templates with rounded settings for practical protocols and quality checks. Includes ramp rate overhead options cycle counts and alerts for unrealistic inputs.

Input Parameters
Target fragment length including any adapters.
Choose a preset or stay on Custom to type your own speed.
Example: 1 means 1 kilobase per minute.
Results round up to practical increments.
Adds time for GC-rich or structured templates.
Extra headroom to improve completion.
Used for total extension time.
Accounts for instrument ramp rates and holds.
What Is PCR Extension?

PCR extension is the phase of a thermal cycling reaction where a DNA polymerase synthesizes new strands by adding nucleotides to a primed template. After denaturation and primer annealing, the reaction is held near the enzyme’s optimum temperature, commonly around 68–72°C, so bases are incorporated in the 5′→3′ direction. Extension time depends mainly on amplicon length, polymerase speed, and template complexity such as GC richness or secondary structure. A typical rule of thumb is 30–60 seconds per kilobase with faster engineered enzymes requiring less. Setting an appropriate duration helps maximize yield while minimizing nonspecific products and incomplete extensions. This calculator estimates a suitable time using fragment length, enzyme rate, GC adjustment, and a safety margin, rounding to increments for protocols.

Results

Enter values and click Calculate to see per cycle and total extension times.

Formula

base_sec = (length_bp / 1000) / rate_kb_min × 60

adjusted_sec = base_sec × (1 + gc_adjust/100) × (1 + safety_margin/100)

rounded_sec = ceil(adjusted_sec / round_inc) × round_inc

total_sec = cycles × (rounded_sec + ramp_overhead)

FAQs
How do you calculate extension time?
Extension time is estimated from amplicon length and polymerase speed, then adjusted for GC content and a user defined margin, and rounded to convenient increments.
Which polymerase speed should I choose?
Use the manufacturer’s guidance for your enzyme. As a starting point, many standard enzymes are ~1 kb/min while engineered high fidelity or ultrafast enzymes can be 2–4 kb/min or more.
How does GC rich content affect timing?
GC rich or structured templates may slow progression. Adding a percentage increase to the time provides headroom to complete difficult regions without overly lengthening easy targets.
When should I add a safety margin?
Use a margin when cycling conditions are uncertain, with new primer pairs, or when targeting long fragments. A modest 5–15% typically balances completion and total run time.
What does rounding accomplish?
Instruments accept whole seconds, and users prefer clean numbers. Rounding up to a set increment ensures adequate time and easier programming without risking incomplete extension.
Should I include adapters in the length?
If adapters or barcodes are part of the sequence being copied, include them. The extension step cares about total base pairs synthesized per cycle.
Can I enter seconds per kilobase instead?
Convert by dividing 60 by your seconds per kilobase value to get kilobases per minute. For example, 30 s/kb corresponds to 2 kb/min.
Does this include denaturation and annealing times?
No. This tool focuses on the extension step. The ramp overhead field can approximate instrument delays, but denaturation and annealing must be configured separately.

Tip: For very long targets consider a two step protocol with high extension temperature and a high speed enzyme validated for long range amplification.

Important Note: All the Calculators listed in this site are for educational purpose only and we do not guarentee the accuracy of results. Please do consult with other sources as well.